Translt lt1

We generated a RV construct (RV-Syn-GTR) consisting of a Murine Moloney Leukemia virus-based RV vector containing a human Synapsin1 promoter driving GFP, the avian sarcoma leukosis virus receptor TVA and RbV glycoprotein (Rgp). To make pSyn-eGFP-TVA-Rgp-WPRE (pSyn-GTR), the GTR DNA fragment was obtained by PCR from the pBOB-SynP-HTB template (a gift from Edward Callaway, Salk Institute, La Jolla, CA) and used to replace GFP in pCAG-GFP-WPRE (a gift from Fred Gage, Salk Institute). A control RV (con-RV) was generated by deleting Rgp from pSyn-GTR. Pseudotyped RV stocks were produced by co-transfecting RV constructs with VSV-G plasmid into the GP2-293 packaging cell line (Clontech, CA). Harvested supernatant containing RV was filtered through a 0.45 μM filter (Gelman Sciences, MI) and concentrated by ultracentrifugation. Titers ranged from 2–5 × 10⁸ cfu/mL. An mCherry (mCh)-expressing, avian envelope glycoprotein subgroup A (EnvA)-pseudotyped RbV (RbV-mCh) was produced as described previously^{28 (link)} with titers of 2–4 × 10⁵ cfu/mL used. Lentivirus (CAMKIIα-Syp-GFP) was generated by transient transfection of the following plasmids: translt-LT1 (Mirus Bio, Madison, WI), vector, psPAX2 and pMD2.G. Supernatant was harvested 48 hours later, filtered and concentrated by ultracentrifugation.

We obtained the luciferase wild-type (WT) and mutant constructs of the pri-miR-21 promoter from Dr. Heike Allgayer^{21 (link)}. Briefly, mutations of AP-1 are at positions −59/−52 bp (AP-1-I), −166/−159 bp (AP-1-II), −225/−220 bp (AP-1-III), and at −656/−663 (AP-1-IV). We used two mutant constructs, one containing AP-1 mutations at AP-1/I, AP-1/II and AP-1/III, and the second containing all AP-1 mutations (AP-1/I – AP-1/IV) and one NF-kappa B mutation at −209/−211. Five hundred thousand cells/well were seeded in 12-well plates overnight at 37° C, 5% CO₂ in complete medium. Next day, transfections were performed according to reagent, TranslT-LT1 (Mirus Bio LLC) instructions. Co-transfection of each Firefly luciferase construct with a Renilla Luciferase control reporter vector (Promega) was done in replicas of three. Plasmid control for the pri-miR-21 promoter constructs was pGL3-Basic. Next day after transfection, 200nM of hr-S100P in complete medium was added to the cells and thereof every 24hrs for 48hrs. Cell homogenization and luciferase assays were performed according to Dual-Luciferase Reporter Assay System kit’s instructions (Promega). Luciferase activity was measured with Sirius Luminometer. Experiments were performed in triplicates.